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Growth Curve for C.acetobutylicum
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EURITY CLASSIFICATION OF THIS PAGE _,,R! PM DOCUMENTATION PAGE
- a 1lb RESTRIV PARIGEAD-A203 242 QTE NJ fILE-AD -A203 23 DISTRIBUTION/AVAILABILITY OF REPORT%0C..^i:IIILATIONj DOWNG SCHEDULE Un 1 imi tedNA ,:
4. PERFORMING ORGANIZATION REPORT NUMB 5. MONITORING ORGANIZATION REPORT NUMBER(S)NAU NA
6a. NAME OF PERFORMING ORGANIZATION 6b OFFICE SYMBOL 7a NAME OF MONITORING ORGANIZATIONHarvard University (if appOke, Office of Naval Research
I NA6c. ADDRESS (City, State, and ZIPCode) 7b ADDRESS (City, State, and ZIP Code)
Division of Applied Sciences 800 N. Ouincy Street29 Oxford Street Arlington, VA 22217-5000Cambridge, MA 02138
Ba. NAME OF FUNDING/SPONSORING 8b OFFICE SYMBOL 9 PROCUREMENT INSTRUMENT IDENTIFICATION NUMBERORGANIZATION (If a cable)Office of Naval Research N00014-88-K-0121
Sc ADDRESS(City, State, and ZIP Code) 10 SOURCE OF FUNDING NUMBERSPROGRAM PROJECT TAS WORK UNI800 N. Ouincy Street ELEMENT NO NO NO00014- ACCESSION
Arlington, VA 22217-5000 88-K-0121 NA11 TITLE (Include Security Classification)
The Role of Microorganisms in Marine Corrosion
12 PERSONAL AUTHOR(S)R. Mitchell
13a TYPE OF REPORT 13b TIME COVERED 14 DATE OF REPORT (Year, Month, Day) 15 PAGE COUNTAnnual FROM 12/1/87TO 11/30/ 8 12/15/88 4
16 SUPPLEMENTARY NOTATION
NA
17 COSATI CODES 18 SUBJECT TERMS (Continue on reverse if necessary and identify by block number)FIELD GROUP SUB-GROUP
Corrosion, bacterial, marine
19 ABSTRACT (Continue on reverse if necessary and identify by block number)
A miniature Devanathan cell probe was modified to grow biofilmsof anaerobic bacteria on palladium surfaces. Hydrogen permeationinto the palladium was detected by measurement of the currentpassing through the cell. Permeation of the gas paralleled thegrowth curve of the bacteria adhering to the palladium surface.Bacteria in the liquid medium contribute minimally to hydrogenpermeation. Very small populations of anaerobic bacteria growingon the palladium surface are capable of contributing significantquantities of hydrogen gas to the metal. ...... : ,NAE A
20 DISTRIBUTION/AVAILABILITY OF ABSTRACT 21 ABSTRACT SECURITY CLASSIFICATION1UNCLASSIFIED/UNLIMITED 0l SAME 4S RPT [- DTIC USERS (U)
22a NAME OF RESPONSIBLE INDIVIDUAL 22b. TELEPHONE (Include Area Code) 22c OFFICE SYMBOLDr. M. Marron (202) 696-4760 ONR
DD FORM 1473,84 MAR 83 APR edition may be used until exhausted SECURITY CLASSIFICATION OF THIS PAGEAll other editions are obsolete
ANNUAL REPORT: Contract N00014-88-K-0121
PRINCIPAL INVESTIGATOR: Ralph Mitchell
CONTRACTOR: Harvard University
CONTRACT TITLE: The Role of Microorganisms inMarine Corrosion
PERIOD OF PERFORMANCE: 12/1/87 - 11/30/88
RESEARCH OBJECTIVES:
To study the role of bacteria in marine corrosion processes.Special emphasis is being placed on the activities of hydrogenproducing bacteria in embrittlement of metals.
PROGRESS:
In our previous research under this contract we have grown purecultures of anaerobic bacteria on mild steel surfaces ofminiature Devanathan cells. Hydrogen gas was produced by thebacteria, and permeated the steel surface of the cell. Thehydrogen permeation caused a rise in the current passing throughthe cell of approximately 8 to 11 ua. Substantial amounts ofacetic and butyric acids were also produced by the bacterium.
During the past year we have developed a more quantitativeassessment of the rate of permeation of evolved bacterialhydrogen gas through metal surfaces. Palladium was substitutedfor steel because of its strong absorption efficiency which isusually as high as 97%.
Figure 1 illustrates a typical growth curve of Clostridiumacetobutylicum. After inoculation there is a lag phase of 8-10hours followed by an exponential growth phase lastingapproximately 3 hours, followed by stationary phase andsporulation. There is a corresponding decrease in the opencircuit potential from approximately -300 millivolts to -500millivolts (Fig. 1A). After 17 hours the hydrogen permeationcurrent begins to increase exponentially (r2 = 0.94) to 127 uAcm then rapidly decreases to baseline rates (Fig. IB).
Efficiency of transport of hydrogen under these conditions wastested by electrochemically charging the i~put side of themembrane over the current range 18 - 176 uA cm . Absorption
2efficiency was found to be 93% over tht range of currents tested(r = 99.99). Therefore 48.6 x 10 - mol HO were produced bybacteria closely associated with the surface of the membrane.Counts of bacteria on th pal]adi z surface were surprisinglylow, averaging less than 10% cells cm It is probable that theabsorbed hydrogen was produced by bacteria closely associatedwith the metal surface. This was confirmed in experiments inwhich 0.1um membranes were placed between the palladium and thebiofilms, preventing adhesion of bacteria to the metal.Permeation currents were reduced dramatically indicating theabsence of hydrogen absorption.
I- 200 A
> - 3002 5 .0 II
0 -400- 1.0 a{J -0.5 q
INOC.
-500. 10.1
B300-
I-Z 200-
o 100
10 20 30 40 s0
TIME (HOURS)C
Figure 1A: Typical open circuit potential characteristics ofpalladium during growth of Clostridium acetobutylicum. Growthmeasured as optical density at 440nm.
Figure IB: Corresponding hydrogen permeation transient.
i. : t ! 5 ... ai ,,3 idel _ .L
3Use of the biologically-adapted Devanathan cell enables us tostudy hydrogen dynamics. Nutrients, gases and bacterial numbersare being continuously monitored and related to the hydrogenpermeation current. Co-cultures, tri-cultures and consortia oforganisms are being studied within the system to understand thecomplex dynamics of mixed populations. Within a mixed microbialfilm the quantity of hydrogen absorbed by a metal is likely to bedifferent from the total hydrogen produced within the film. Themost important factor is the probability of hydrogen consumptionby other bacteria growing on the metal surface. Hydrogenembrittlement may be determined by the outcome of competition forhydrogen between the metal and hydrogen-consuming bacteria.
OBJECTIVES FOR THE NEXT YEAR:
We plan to continue to use conventional electrochemicaltechniques for hydrogen permeation measurement through definedmetal foils. We will continue to quantify hydrogen permeationwith pure cultures of bacteria using the defined system, allowingcalculations of hydrogen production on a per cell basis.Comparison will then be made with co- and mixed cultures andinterspecific hydrogen transfer investigated. Effect ofmetabolites (e.g., sulfide, organic acids, etc.) will also bestudied using the defined system. Once these reactions have beenquantified with palladium, the work will extend to thin foils ofmetals susceptible to embrittlement, particularly high strengthsteels. Hydrogen permeation through these steels will becalibrated electrochemically. Permeation data will besupplemented with stress testing of steels after exposure tobacterial cultures and subsequent microstructural analysis of themetal.
PUBLICATIONS AND RESEARCH ABSTRACTS
1. Ford, T.E., M. Walch and R. Mitchell, 1987, Corrosion ofmetals by thermophilic microorganisms, Materials Performance26:35-39.
2. Ford, T.E., J.S. Maki and R. Mitchell, 1987, The role ofmetal-binding bacterial exopolymers in corrosion processes,CORROSION/87, Paper No. 380, Natl. Assoc. Corrosion Eng.,Houston, Texas.
3. Black, J.P., T.E. Ford, J.S. Maki and R. Mitchell, 1987,Processes of metal deposition by Pedomicrobium exopolymers.American Society for Microbiology Annual Meeting, Atlanta,Georgia.
44. Black, J.P., T.E. Ford and R. Mitchell, 1988, Corrosionbehavior of metal-binding exopolymers from iron- andmanganese- depositing bacteria, CORROSION/88, Paper No. 94,Natl. Assoc. Corrosion Eng., Houston, Texas.
5. Ford, T.E., J.P. Black, E. Reger and R. Mitchell, 1988,Metal-binding by exopolymers of surface bacteria, AmericanSociety for Microbiology Annual Meeting, Miami, Florida.
6. Ford, T.E., M. Walch, R. Mitchell, M.J. Kaufman, J.R.Vestal, S.A. Ditner, and M.A. Lock, 1989, Microbial filmFormation on metals in an enriched arctic river, Biofouling(In Press).
7. Ford, T.E., J.S. Maki and R. Mitchell, 1989, Involvement ofbacterial exopolymers in biodeterioration of metals,BIODETERIORATION 7, The Biodeterioration Society, Cambridge,UK, (In Press).
8. Walch, M., T.E. Ford and R. Mitchell, 1989, Influence ofhydrogen-producing bacteria on hydrogen uptake by steel,Corrosion (In Press).
9. Ford, T.E. and R. Mitchell, 1989, Hydrogen embrittlement: amicrobiological perspective, CORROSION/89, Paper No. 189,Natl. Assoc. Corrosion Eng., Houston, Texas (In Press).
INVENTIONS:
None
TRAINING ACTIVITIES:
One graduate student and one undergraduate are working on theproj ec t.
WOMEN AND MINORITIES:
One
NON-CITIZENS:
One
AWARDS:
None